[Site-directed mutagenesis and function analysis of glgC gene from Escherichia coli.].
Using genomic DNA of Escherichia coli JM109 as a template, glgC gene was amplified by polymerase chain reaction (PCR). The full coding sequence of this gene is 1296 bp. To get 3 mutants that amino acids changed: P295S (V121A, M151I, V334D), G336D and P295S/G336D (K109R) by recombinant PCR, respectively named 295+3, 336 and 295/336+1. The 3 mutants and the original glgC were subcloned into the prokaryotic expression vector pET-32a, and these recombinant expression plasmids were transformed into E. coli BL21 (DE3) for effective expression. The host cells were induced with IPTG and then identified by SDS-PAGE. A specific fused-expression product 67 kDa was detected, which was the same as the deduced protein. In the host cells above, the biological activities of the expressed products were detected by iodine vapor staining and glycogen content testing. The host cell transformed with the mutated gene-336 had higher glycogen content, which was identical to the gene-295/336+1. This confirmed that Pro295Ser could not reinforce the decrease of the feedback inhibition effect of the AGPase. Meanwhile, another host cell transformed with the mutated gene-295+3 showed decreased glycogen rather than the expected increasing glycogen. This might be caused by another mutation, Val334Asp in gene-295+3, which might induce the change of the allosteric region of the objective protein.
Zhou
Shuang
S
Key Laboratory of Resource Biology & Eco-environment, Ministry of Education, Sichuan Key Laboratory of Molecular Biology & Bio-technology, College of Life Science, Sichuan University, Chengdu 610065, China E-mail: mxxhappygirl@163.com.
Xu
Ke
K
He
Ming-Xiong
MX
Zhang
Yi-Zheng
YZ
D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction